Connective Tissue Growth Factor is a Target of Notch Signaling in Cells of the Osteoblastic Lineage

Abstract

Connective tissue growth factor (Ctgf) or CCN2 is a protein synthesized by osteoblasts necessary for skeletal homeostasis, although its overexpression inhibits osteogenic signals and bone formation. Ctgf is induced by bone morphogenetic proteins, transforming growth factor β and Wnt; and in the present studies, we explored whether Notch regulated Ctgf expression in osteoblasts. We employed RosaNotch mice, where the Notch intracellular domain (NICD) is expressed following the excision of a STOP cassette, placed between the Rosa26 promoter and NICD. Notch was activated by transduction of adenoviral vectors expressing Cre recombinase (Ad-CMV-Cre). Notch induced Ctgf mRNA levels in a time dependent manner and increased Ctgf heterogeneous nuclear RNA. Notch also destabilized Ctgf mRNA shortening its half-life from 13 h to 3 h. The effect of Notch on Ctgf expression was lost following Rbpjκ downregulation, demonstrating that it was mediated by Notch canonical signaling. However, downregulation of the classic Notch target genes Hes1, Hey1 and Hey2 did not modify the effect of Notch on Ctgf expression. Wild type osteoblasts exposed to immobilized Delta-like 1 displayed enhanced Notch signaling and increased Ctgf expression. In addition to the effects of Notch in vitro, Notch induced Ctgf in vivo, and calvariae and femurs from RosaNotch mice mated with transgenics expressing the Cre recombinase in cells of the osteoblastic lineage exhibited increased expression of Ctgf. In conclusion, Ctgf is a target of Notch canonical signaling in osteoblasts, and may act in concert with Notch to regulate skeletal homeostasis.

Smooth muscle cell-specific Notch1 haploinsufficiency restricts the progression of abdominal aortic aneurysm by modulating CTGF expression

“…Previous studies have demonstrated that Notch1 signaling directly regulates CTGF in osteoclasts which increases fibrotic response[53]. To determine whether CTGF functionally modulates a shift towards a synthetic phenotype, we performed collagen gel contraction assay using VSMCs from the abdominal aortae of Apoe -/- and Notch1 +/- ;Apoe -/- mice.…”

Parathyroid hormone inhibits Notch signaling in osteoblasts and osteocytes

“…Although Jag1 has a documented skeletal function, Dll1 was chosen because it enhances Notch activation consistently in multiple cell lineages and in osteoblasts, and both ligands induce Notch target genes to a comparable extent (Canalis and Zanotti unpublished observations) [49–51]. A PTH dose of 10 nM, known to have a maximal effect on osteoblast function was used for the present studies [52–55].…”

The lateral meningocele syndrome mutation causes marked osteopenia in mice

“…Cells from the last 3 digestions were pooled and seeded at a density of 10,000 cells/cm 2 , as previously described as previously reported (50,68). Osteoblast-enriched cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with non-essential amino acids (both from Thermo Fisher Scientific), 20 mM HEPES, 100 µg/ml ascorbic acid (both from Sigma-Aldrich) and 10% heat-inactivated fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA) in a humidified 5% CO2 incubator at 37C.…”